Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics.

OBJECTIVES: To establish and forensically verify a 42 microhaplotypes (mircohaps, MHs) multiplex assay system based on next-generation sequencing (NGS), and to explore the application value of this system in the practice of forensic genetics. METHODS: A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGxTM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated. RESULTS: The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo (≤2 500 ng/µL), humic acid (≤9 ng/µL), hemoglobin(≤20 µmol), and urea (≤200 ng/µL) and to detect mixtures of 2 people (1∶19), 3 people (1∶1∶9) and 4 people (1∶1∶1∶9). Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10-30 and 1-3.77×10-11, respectively, and the average effect value of alleles was 2.899. CONCLUSIONS: The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Circadian clock1a coordinates neutrophil recruitment via nfe212a/duox-reactive oxygen species pathway in zebrafish.

Neutrophil recruitment to inflammatory sites appears to be an evolutionarily conserved strategy to fight against exogenous insults. However, the rhythmic characteristics and underlying mechanisms of neutrophil migration on a 24-h timescale are largely unknown. Using the advantage of in vivo imaging of zebrafish, this study explored how the circadian gene clock1a dynamically regulates the rhythmic recruitment of neutrophils to inflammatory challenges. We generated a clock1a mutant and found that neutrophil migration is significantly increased in caudal fin injury and lipopolysaccharide (LPS) injection. Transcriptome sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporting experiments suggest that the clock1a gene regulates neutrophil migration by coordinating the rhythmic expression of nfe212a and duox genes to control the reactive oxygen species (ROS) level. This study ultimately provides a visual model to expand the understanding of the rhythmic mechanisms of neutrophil recruitment on a circadian timescale in a diurnal organism from the perspective of ROS.

Front-face excitation-emission matrix fluorescence spectroscopy combined with interpretable deep learning for the rapid identification of the storage year of Ningxia wolfberry.

Ningxia wolfberry stored for many years may be disguised as fresh wolfberry by unscrupulous traders and sold for huge profits. In this work, the front-face excitation-emission matrix (FF-EEM) fluorescence spectroscopy coupled with interpretable deep learning was proposed to identify the storage year of Ningxia wolfberry in a lossless, fast and accurate way. Alternating trilinear decomposition (ATLD) algorithm was used to decompose the three-way data array obtained by Ningxia wolfberry samples, extracting the chemically meaningful information. Meanwhile, a convolutional neural network (CNN) model for the identification of the storage year of Ningxia wolfberry, called EEMnet, was proposed. The model successfully classified wolfberry samples from different storage years by extracting the subtle feature differences of the spectra, and the correct classification rate of the training set, test set and prediction set was more than 98%. In addition, a series of interpretability analyses were implemented to break the "black box" of the deep learning model. These results indicated that the method based on FF-EEM fluorescence spectroscopy combined with EEMnet could quickly and accurately identify the year of Ningxia wolfberry in a green way, providing a new idea for the identification of the storage years of Chinese medicinal materials.

Protein Requirements of Oncorhynchus mykiss Cultured in the Convection-Water Cages by Evaluating Growth, Body Composition and Liver Health.

The diet formulation for trout has changed dramatically over the last decade due to changes in the ingredient markets and advances in feed processing technology. The protein requirements of Oncorhynchus mykiss were established at the end of the last century, and it is unclear whether these requirements are applicable to modern dietary formulations. Therefore, an eight-week feeding trial was performed to measure the protein requirements of O. mykiss by evaluating growth, body composition, antioxidation property, innate immune response and liver morphology. The five experimental diets were prepared to contain the same levels of crude lipid (120 g/kg) and graded levels of crude protein (356.3, 383.9, 411.5, 439.2 and 466.8 g/kg). The results suggested that the growth, feed utilization and whole-body crude protein levels were significantly increased when fish were fed diets containing 439.2 and 466.8 g/kg crude protein. Meanwhile, low dietary protein levels (356.3 and 383.9 g/kg) significantly down-regulated the mRNA levels of insulin-like growth factor I, catalase, glutathione peroxidase, superoxide dismutase, complement 3 and lysozyme, and also up-regulated the insulin-like growth factor binding protein 1 as well as proinflammatory cytokine expression in the liver, including interleukin 1ß, interleukin 8 and tumor necrosis factor-α. Moreover, low dietary protein levels (356.3 and 383.9 g/kg) damaged liver structure, suppressed total antioxidative capacity and increased the malondialdehyde content in liver. In conclusion, high dietary protein (439.2 and 466.8 g/kg) promoted fish growth, while low dietary protein (356.3 and 383.9 g/kg) damaged liver structure, induced oxidative stress and inflammatory responses and weakened non-specific immunity. The protein requirement of O. mykiss reared in the convection-water cages is no less than 439.2 g/kg for optimal growth, antioxidant and immune properties.

Ophthalmic Solution of Smart Supramolecular Peptides to Capture Semaphorin 4D against Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision loss in working age population. Intravitreal injection of anti-VEGF antibody is widely used in clinical practice. However, about 27% of patients show poor response to anti-VEGF therapy and about 50% of these patients continue to have macular thickening. Frequent intravitreal injections of antibody may increase the chance of endophthalmitis and cause visual loss or even blindness once happened. Therefore, there is a greatly urgent need for novel noninvasive target to treat DR clinically. Here, the formulation of a smart supramolecular peptide (SSP) eye drop for DR treatment that is effective via specifically identifying and capturing soluble semaphorin 4D (sSema4D), a strongly pro-angiogenesis and exudates factor, is reported. The SSP nanostructures encapsulate sSema4D so that all biological effects mediated by three receptors of sSema4D are inhibited, thereby significantly alleviating pathological retinal angiogenesis and exudates in DR. Moreover, it is found that combination of SSPs eye drop and anti-VEGF injection shows better therapeutic effect over anti-VEGF treatment alone. Overall, SSP eye drop provide an alternative and effective method for noninvasive treatment for DR.

Intelligent analysis of excitation-emission matrix fluorescence fingerprint to identify and quantify adulteration in camellia oil based on machine learning.

Camellia oil (CAO) is a premium edible vegetable oil with medical value and biological activity, but it is susceptible to adulteration. Therefore, the demand for intelligent analysis to decipher the category and proportion of adulterated oil in CAO was the main driver of this work. Excitation-emission matrix fluorescence (EEMF) spectra of 933 vegetable oil samples were characterized by a chemometric method to obtain chemically meaningful information. Authenticity identification models were constructed using four machine learning methods to realize the discrimination of oil species adulterated in CAO mixtures. Meanwhile, quantitative models were established aiming at the fraud of CAO proportion in blended oil. Results showed that the specially constructed CNN obtained the optimal performance when evaluating unseen real-world samples, with a classification accuracy of 95.8% and 92.2%, and mean-absolute quantitative errors between 2.6 and 6.7%. Therefore, EEMF fingerprints coupled with machine learning are expected to provide intelligent and accurate analysis for authenticity detection of CAO.

Chemometrics-enhanced high-performance liquid chromatography-diode array detection strategy to quantify protoberberine alkaloids in varying Coptidis Rhizoma-related medicines.

Quantitation of protoberberine alkaloids is an essential guarantee for efficacy control and medication safety of Coptidis Rhizoma (CR) related medicines. Traditional univariate chromatography faced challenges with co-elution, unknown interferences, and retention time shift when analyzing isomeric analytes in varying sample matrices. We presented a chemometrics-enhanced high-performance liquid chromatography-diode array detection (HPLC-DAD) strategy for simultaneous quantification of six protoberberine alkaloids and processed multi-channels chromatographic-spectral data with four second-order calibration algorithms. Chromatographic conditions were firstly optimized. Four groups of predicted samples were modeled individually with the designed calibration set. Mathematical resolutions were then obtained, and pseudo-univariate regression gave the quantitative concentration of each analyte. Four models were scored on fit, linearity, recovery, and robustness, where alternating trilinear decomposition assisted multivariate curve resolution (ATLD-MCR) exhibited an optimal and stable performance. Besides, the resolved spectra presented high consistency with the actual spectra (r≥0.9993). Limits of quantification (LOQ) fully met the pharmacopoeia stipulation and were 0.17, 0.60, 0.19, 0.74, 0.15, and 0.38 µg mL-1 for columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine, respectively. The importance of this strategy is to exploit collinearity resolution and additional selectivity that permit accurate quantitation at poor chromatographic resolutions, avoiding individual pretreatment and HPLC optimizations for different samples. This study provides a universal alternative for routine quality assessment of protoberberine alkaloids in CR-related medicines.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Fluoxetine modifies circadian rhythm by reducing melatonin content in zebrafish.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), increases the serotonin levels in the brain to treat depression. Antidepressants have been demonstrated to modulate circadian rhythm, but the underlying mechanisms by which antidepressants regulate circadian rhythm require more research. This study aimed to investigate the role of FLX on circadian rhythm by analyzing the movement behavior and internal circadian oscillations in zebrafish. The results showed that the expression of clock genes clock1a and bmal1b was significantly down-regulated, and the amplitude reduction and phase shift were observed after FLX treatment. Furthermore, FLX exposure inhibited the expression of aanat2, which led to a decrease in nocturnal melatonin secretion. aanat2-/- larvae showed disrupted circadian rhythm. These findings may help reveal the effect of FLX exposure on the circadian rhythm and locomotor activity. It may provide theoretical data for the clinical application of FLX.

The influences and regulatory mechanisms of magnetic fields on circadian rhythms.

A variety of devices used in daily life and biomedical field will generate magnetic fields with different parameters, raising concern about their influences on people's physiological functions. Multiple experimental works have been devoted to the influences of magnetic fields on circadian rhythms, yet the findings were not always consistent due to the differences in magnetic field parameters and experimental organisms. Also, clear regulatory mechanisms have not been found. By systematizing the major achievements in research on magnetic and circadian rhythms based on magnetic flux density and analyzing the potential mechanisms of the magnetic fields affecting circadian rhythms, this review sheds light on the effects of magnetic fields on circadian rhythms and the potential applications in biomedicine.

Muscarinic acetylcholine receptors regulate inflammatory responses through arginases 1/2 in zebrafish.

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in various effector cells and have been proved to play vital roles in smooth muscle contraction and digestive secretion. However, there are relatively few literatures revealing the roles of mAChRs in inflammatory processes, and its underlying regulatory mechanisms have not been elucidated. Taking the advantages of live imaging of zebrafish, we found that inhibition of mAChRs resulted in increased neutrophils recruitment and proinflammatory cytokines expression, whereas activation of mAChRs led to opposite outcome. Subsequently, we found that mAChRs regulated the expression of arginases (args), and pharmacological intervention of args level could reverse the effects of mAChRs on neutrophils migration and cytokines expression, suggesting that args are important downstream proteins of mAChRs that mediate the regulation of inflammatory response. In this study, we identified args as novel downstream proteins of mAChRs in inflammatory responses, providing additional evidence for system immune regulation of cholinergic receptors.

Hypoxia regulates cytokines expression and neutrophils migration by ERK signaling in zebrafish.

Normal dissolved oxygen in water is essential for maintaining the physiological functions of fish, but environmental pollution, such as eutrophication can lead to a decrease in oxygen content in water. How this reduction of dissolved oxygen in water affects the immune functions of fish and the potential regulatory mechanisms have not been thoroughly elucidated. In this study, we made full use of the aquatic model animal zebrafish to explore this question. In a model of LPS-induced inflammation, we found that hypoxia induced by infusing nitrogen into water increased the expression of pro-inflammatory cytokines, such as il-1ß, il-6, and il-8. In vivo imaging also showed that hypoxia significantly increased neutrophil migration to the site of caudal fin injury in the transgenic line. Subsequently, we found that the phosphorylation level of ERK protein was significantly activated upon hypoxia and proved the roles of ERK signaling in the expression of pro-inflammatory cytokines and neutrophil migration in zebrafish. This study indicated that reduced water oxygen significantly increases the inflammatory response of the zebrafish.

N-acetylcholine receptors regulate cytokines expression and neutrophils recruitment via MAPK/ERK signaling in zebrafish.

N-acetylcholine receptors (AChRs) are mainly distributed in the postsynaptic membrane and have been widely studied for their control of muscle contraction by regulating neural action potentials. However, the influences of AChRs on immune responses and potential mechanisms remain unclear. Here, we used the advantages of live imaging of zebrafish to explore the regulation process of AChRs on inflammatory responses. Pharmacologically activating of the receptor, we found that the expression of pro-inflammatory cytokines il-1ß, il-6, tnf-α and il-8 was significantly up-regulated and neutrophil migration to injury sites was also significantly increased. However, these phenomena were reversed under antagonism of the receptor activity. Results showed that interfering with nAChRs functions did not significantly affect zebrafish motion behavior. Results also showed that activation and antagonism of nAChRs function could regulate the phosphorylation of ERK protein respectively. We further demonstrated that ERK participated in the regulation of AChRs in cytokines expression and neutrophils migration in zebrafish. This study preliminarily revealed the roles of AChRs in inflammatory processes and their potential mechanism, providing additional evidence of peripheral immune regulation by cholinergic receptors.

Dysfunction of the Blood-brain Barrier in Cerebral Microbleeds: from Bedside to Bench.

Cerebral microbleeds (CMBs) are a disorder of cerebral microvessels that are characterized as small (<10 mm), hypointense, round or ovoid lesions seen on T2*-weighted gradient echo MRI. There is a high prevalence of CMBs in community-dwelling healthy older people. An increasing number of studies have demonstrated the significance of CMBs in stroke, dementia, Parkinson's disease, gait disturbances and late-life depression. Blood-brain barrier (BBB) dysfunction is considered to be the event that initializes CMBs development. However, the pathogenesis of CMBs has not yet been clearly elucidated. In this review, we introduce the pathogenesis of CMBs, hypertensive vasculopathy and cerebral amyloid angiopathy, and review recent research that has advanced our understanding of the mechanisms underlying BBB dysfunction and CMBs presence. CMBs-associated risk factors can exacerbate BBB breakdown through the vulnerability of BBB anatomical and functional changes. Finally, we discuss potential pharmacological approaches to target the BBB as therapy for CMBs.

Mfsd2a overexpression alleviates vascular dysfunction in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the common complications in diabetic patients. Nowadays, VEGF pathway is subject to extensive research. However, about 27% of the patients have a poor visual outcome, with 50% still having edema after two years' treatment of diabetic macular edema (DME) with ranibizumab. Docosahexaenoic acid (DHA), the primary ω-3 long-chain polyunsaturated fatty acid (LC-PUFA), reduces abnormal neovascularization and alleviates neovascular eye diseases. A study reported that fish oil reduced the incidence of retinopathy of prematurity (ROP) by about 27.5% in preterm infants. Although ω-3 LC-PUFAs protects against pathological retinal neovascularization, the treatment effectiveness is low. It is interesting to investigate why DHA therapy fails in some patients. In human vitreous humor samples, we found that the ratio of DHA and DHA-derived metabolites to total fatty acids was higher in vitreous humor from DR patients than that from macular hole patients; however, the ratio of DHA metabolites to DHA and DHA-derived metabolites was lower in the diabetic vitreous humor. The expression of Mfsd2a, the LPC-DHA transporter, was reduced in the oxygen-induced retinopathy (OIR) model and streptozotocin (STZ) model. In vitro, Mfsd2a overexpression inhibited endothelial cell proliferation, migration and vesicular transcytosis. Moreover, Mfsd2a overexpression in combination with the DHA diet obviously reduced abnormal retinal neovascularization and vascular leakage, which is more effective than Mfsd2a overexpression alone. These results suggest that DHA therapy failure in some DR patients is linked to low expression of Mfsd2a, and the combination of Mfsd2a overexpression and DHA therapy may be an effective treatment.

Immune Cells in the BBB Disruption After Acute Ischemic Stroke: Targets for Immune Therapy?

Blood-Brain Barrier (BBB) disruption is an important pathophysiological process of acute ischemic stroke (AIS), resulting in devastating malignant brain edema and hemorrhagic transformation. The rapid activation of immune cells plays a critical role in BBB disruption after ischemic stroke. Infiltrating blood-borne immune cells (neutrophils, monocytes, and T lymphocytes) increase BBB permeability, as they cause microvascular disorder and secrete inflammation-associated molecules. In contrast, they promote BBB repair and angiogenesis in the latter phase of ischemic stroke. The profound immunological effects of cerebral immune cells (microglia, astrocytes, and pericytes) on BBB disruption have been underestimated in ischemic stroke. Post-stroke microglia and astrocytes can adopt both an M1/A1 or M2/A2 phenotype, which influence BBB integrity differently. However, whether pericytes acquire microglia phenotype and exert immunological effects on the BBB remains controversial. Thus, better understanding the inflammatory mechanism underlying BBB disruption can lead to the identification of more promising biological targets to develop treatments that minimize the onset of life-threatening complications and to improve existing treatments in patients. However, early attempts to inhibit the infiltration of circulating immune cells into the brain by blocking adhesion molecules, that were successful in experimental stroke failed in clinical trials. Therefore, new immunoregulatory therapeutic strategies for acute ischemic stroke are desperately warranted. Herein, we highlight the role of circulating and cerebral immune cells in BBB disruption and the crosstalk between them following acute ischemic stroke. Using a robust theoretical background, we discuss potential and effective immunotherapeutic targets to regulate BBB permeability after acute ischemic stroke.

Cordycepin inhibits inflammatory responses through suppression of ERK activation in zebrafish.

As a natural extract, cordycepin has been shown to play important regulatory roles in many life activities. In the study, the effects of cordycepin on inflammatory responses and the underlying mechanisms was explored using a zebrafish model. In the model of LPS-induced inflammation, cordycepin was found to significantly inhibited the expression of pro-inflammatory cytokines such as tnf-α, il-1ß, il-6, and il-8. Using in vivo imaging model, cordycepin significantly inhibited fluorescent-labeled neutrophils migrating towards injury sites. Furthermore, results showed that the phosphorylation level of ERK protein dramatically decreased after cordycepin treatment. Meanwhile, the ERK inhibitor, PD0325901, significantly inhibited the expression of pro-inflammatory cytokines in LPS-induced inflammatory model and neutrophils migration in the caudal fin injury model. This study indicated the important roles of cordycepin in inhibiting LPS and injury-induced inflammation and preliminarily explained the role of ERK protein in this process.

Excitation-emission matrix fluorescence spectroscopy coupled with multi-way chemometric techniques for characterization and classification of Chinese lager beers.

This paper proposed excitation-emission matrix fluorescence spectroscopy coupled with multi-way chemometric techniques for characterization and classification of Chinese pale lager beers produced by different manufacturers. The undiluted and diluted beer samples presented different fluorescence fingerprints. Three-way and four-way parallel factor analysis (PARAFAC) were used to decompose the skillfully constructed three-way and four-way data arrays, respectively, to further achieve beer characterization and feature extraction. Based on the features extracted in different ways, four strategies for beer classification were proposed. In each strategy, three supervised classification methods including linear discriminant analysis (LDA), partial least squares discriminant analysis (PLS-DA) and k-nearest neighbor (kNN) were used to build discriminant models. By comparison, PARAFAC-data fusion-kNN method in strategy 3 and four-way PARAFAC-kNN method in strategy 4 obtained the best classification results. The classification strategy based on four-way sample-excitation-emission-dilution level data array was proposed to solve the problem of beer classification for the first time.

A chemometric comparison of different models in fluorescence analysis of dabigatran etexilate and dabigatran.

In this paper, a simple, rapid, low-cost and potential method was established for the simultaneous quantitative analysis of dabigatran etexilate (DABE) and dabigatran (DAB) in spiked biological fluids. It combined excitation-emission matrix fluorescence (EEMF) with different second-order calibration methods, including the self-weighted alternating normalized residue fitting (SWANRF) algorithm based on trilinear decomposition model, the multivariate curve resolution - alternating least-squares (MCR-ALS) based on bilinear decomposition model and the unfolded partial least-square coupled with residual bilinearization (U-PLS/RBL) based on latent variables model. The proposed method showed "second-order advantage", that is, satisfactory quantitative results were successfully obtained even in the presence of unknown interferences and serious spectral overlap. The recoveries of DABE and DAB in spiked biological fluids were 91.7%-101.7% for SWANRF, 95.9%-117.8% for MCR-ALS, 83.0%-109.6% for U-PLS/RBL, respectively. Figures of merit and other statistical parameters were also calculated to assess the performance of the proposed method. Moreover, the modeling procedures and characteristics of three different models in EEMF analysis were discussed and compared.

The role of semaphorins in small vessels of the eye and brain.

Small vessel diseases, such as ischemic retinopathy and cerebral small vessel disease (CSVD), are increasingly recognized in patients with diabetes, dementia and cerebrovascular disease. The mechanisms of small vessel diseases are poorly understood, but the latest studies suggest a role for semaphorins. Initially identified as axon guidance cues, semaphorins are mainly studied in neuronal morphogenesis, neural circuit assembly, and synapse assembly and refinement. In recent years, semaphorins have been found to play important roles in regulating vascular growth and development and in many pathophysiological processes, including atherosclerosis, angiogenesis after stroke and retinopathy. Growing evidence indicates that semaphorins affect the occurrence, perfusion and regression of both the macrovasculature and microvasculature by regulating the proliferation, apoptosis, migration, barrier function and inflammatory response of endothelial cells, vascular smooth muscle cells (VSMCs) and pericytes. In this review, we concentrate on the regulatory effects of semaphorins on the cell components of the vessel wall and their potential roles in microvascular diseases, especially in the retina and cerebral small vessel. Finally, we discuss potential molecular approaches in targeting semaphorins as therapies for microvascular disorders in the eye and brain.